1. Area of the Art
The present invention relates generally to the detection and identification of proteins, as well as various forms and subunits of proteins, which have the potential utility as diagnostic markers. In particular, the present invention relates to the detection or determination of inactive forms of prostate specific antigens in the biological fluid of humans, and methods for the improved detection of prostate cancer.
2. Description of the Related Art
The measurement of serum prostate specific antigen (PSA) is widely used for the screening and early detection of prostate cancer [1–3]. Serum PSA that is measurable by current clinical immunoassays exists primarily as either the free “non-complexed” form (free PSA), or as a complex with □-1antichymotrypsin (ACT) [1–5]. The ratio of free to total PSA in serum has been demonstrated to significantly improve the discrimination of PCa from BPH, with higher ratios correlating with a lower risk of prostate cancer [1–7].
The biological mechanism for the variable levels of free PSA in serum is unknown. The serum PSA that has become complexed is likely to be relatively homogeneous since this represents enzymatically active, intact PSA. The PSA released from the PSA-ACT complex in prostate cancer (PCa) and benign prostate hyperplasia (BPH) serum was found to be indistinguishable from seminal plasma PSA, which confirms this assumption [8]. It follows that free PSA may offer better biochemical insight, and that a characterization of the molecular forms of free PSA could help elucidate their prostatic origin and mechanism of release into the serum.
Various mixtures of PSA containing inactive forms of PSA have been isolated from prostate tissues and seminal plasma. The PSA from BPH tissue has been characterized in a more general way as being more highly internally clipped [9], though no single form or forms of PSA were isolated from the mixture of clipped and unclipped PSA forms studied in this work. PSA in seminal plasma has been separated by chromatographic methods into fractions shown to contain various mixtures of clipped and non-clipped PSA [10]. However, in none of these studies have antibodies been developed to any single form of inactive PSA or have these PSA forms been demonstrated in serum.
In serum, the free (uncomplexed) PSA is now known to be comprised of at least two specific forms of inactive PSA. One form has been identified as the proenzyme, or precursor form of PSA (pPSA), which includes truncated forms of PPSA that are more associated with cancer [11–13]. A second specific form of PSA, called BPSA, is an internally cleaved or degraded form of PSA that is more highly associated with BPH [14;15]. BPSA was isolated and extensively characterized as a homogeneous species of PSA. A detailed review of BPSA and pPSA has been recently published [16].
Another method of describing and measuring sub-populations of free forms of PSA in serum has also been reported by Nurmikko [17]. In this case, intact PSA (PSA-I) is defined as free PSA that is not clipped at Lys145. This assay recognizes free PSA forms that are not internally clipped at Lysine145, the site of the most common form of degraded PSA in seminal plasma. The nature of the PSA measured by this method is not well-defined except that it is not clipped at Lys145. As such, this assay would measure mixtures of pPSA forms in combination with other uncharacterized inactive forms of PSA not clipped at Lys145.
Because the clinically useful concentrations of PSA are present in the serum at less than 10 ng/ml, it is necessary to develop immunoassays or other very sensitive methods to measure the PSA. The current commercial free PSA immunoassays measure the combined sum of all forms of free PSA, levels typically below 2 ng/ml. Antibodies and immunoassays for pPSA, BPSA and PSA-I remain the only reported research immunoassays for measuring sub-forms of free PSA in serum. Therefore, a need exists to develop novel methods for measuring different forms of free PSA, and further study their associations with different prostate disease status.